Self-renewal Capacity Of Hematopoietic Stem Cells Research Articles

The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.

Nynrin Preserves Hematopoietic Stem Cell Function Via the Ppif-Mediated Mitochondrial Permeability Transition

Mitochondria have recently been identified as a critical regulator for homeostasis of hematopoietic stem cells (HSCs). However, the mechanism underlying HSC regulation remains poorly clarified. Here, we identify transcription factor Nynrin as a novel regulator of HSC maintenance through modulation of mitochondrial function. We demonstrate that Nynrin knockout leads to significantly decreased long-term HSC frequency, markedly reduced HSC dormancy and self-renewal capacity in steady-state and stress hematopoiesis. Nynrin-deficient HSCs are more compromised in tolerance of irradiation- and 5-fluorouracil-induced stresses and exhibit typical phenotypes of necrosis. Mechanistically, Nynrin deletion induces transactivation of Ppif, its product cyclophilin D is one of the prominent members of mitochondrial permeability transition (mPT). Overexpression of Ppif exhausts long-term HSC, impairs genotoxic hematopoietic recovery, and enhances mPT, resulting in mitochondrial swelling and reactive oxygen species production. Suppression of Ppif essentially rescues the phenotypic and functional defects in Nynrin-deleted HSCs. Collectively, our data, for the first time, characterize Nynrin as a critical regulator of HSC function acting through the Ppif/ mPT mitochondria axis and highlight the importance of Nynrin in HSC maintenance. These observations provide new insights into the mechanisms for controlling HSC fate.

RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

RhTPO Ameliorates Radiation-Induced Long-Term Hematopoietic Stem Cell Injury in Mice.

Exposure to medium and high doses of ionizing radiation (IR) can induce long-term bone marrow (BM) suppression. We previously showed that recombinant human thrombopoietin (rhTPO) significantly promotes recovery from hematopoietic-acute radiation syndrome, but its effect on long-term BM suppression remains unknown. C57BL/6 mice were exposed to 6.5 Gy γ-rays of total body irradiation (TBI) at a dose-rate of 63.01 cGy per minute, and the mice were treated with rhTPO (100 μg; intramuscular injection) or vehicle at 2 h after TBI. All mice were killed one or two months after TBI for analysis of peripheral blood cell counts, long-term hematopoietic stem cell (HSC) frequency, and BM-derived clonogenic activity. The HSC self-renewal capacity was analyzed by BM transplantation. The levels of reactive oxygen species (ROS) production and ratios of γH2AX+ and p16, p53, and p21 mRNA in HSCs were measured by flow cytometry and real-time polymerase chain reaction, respectively. Treatment with rhTPO reduced long-term myelosuppression by improving long-term hematopoietic reconstitution (p < 0.05) after transplantation and resting state maintenance of HSCs (p < 0.05). Moreover, rhTPO treatment was associated with a sustained reduction in long-term ROS production, reduction of long-term DNA damage, diminished p53/p21 mRNA expression, and prevention of senescence after TBI. This study suggests rhTPO is an effective agent for treating IR-induced long-term BM injury because it regulates hematopoietic remodeling and HSC cycle disorder through the ROS/p53/p21/p16 pathway long term after IR.

Aging and Clonal Behavior of Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are the only cell population that possesses both a self-renewing capacity and multipotency, and can give rise to all lineages of blood cells throughout an organism’s life. However, the self-renewal capacity of HSCs is not infinite, and cumulative evidence suggests that HSCs alter their function and become less active during organismal aging, leading ultimately to the disruption of hematopoietic homeostasis, such as anemia, perturbed immunity and increased propensity to hematological malignancies. Thus, understanding how HSCs alter their function during aging is a matter of critical importance to prevent or overcome these age-related changes in the blood system. Recent advances in clonal analysis have revealed the functional heterogeneity of murine HSC pools that is established upon development and skewed toward the clonal expansion of functionally poised HSCs during aging. In humans, next-generation sequencing has revealed age-related clonal hematopoiesis that originates from HSC subsets with acquired somatic mutations, and has highlighted it as a significant risk factor for hematological malignancies and cardiovascular diseases. In this review, we summarize the current fate-mapping strategies that are used to track and visualize HSC clonal behavior during development or after stress. We then review the age-related changes in HSCs that can be inherited by daughter cells and act as a cellular memory to form functionally distinct clones. Altogether, we link aging of the hematopoietic system to HSC clonal evolution and discuss how HSC clones with myeloid skewing and low regenerative potential can be expanded during aging.

Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion

AbstractHow genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1−/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1−/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Haploid Kmt2c Deletions Enhance Hematopoietic Stem Cell Mobilization in Response to Granulocyte-Colony Stimulating Factor

KMT2C is one of several tumor suppressor genes deleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as part of larger chromosome 7q deletions. These deletions are particularly common in therapy-related MDS/AML, raising the question of whether loss of one or more 7q genes conveys a selective advantage to hematopoietic stem cells (HSCs) in the setting of chemotherapy-induced stress. We recently showed that haploid Kmt2c deletions do indeed enhance HSC self-renewal capacity. However, the deletions do not drive HSCs into cycle; instead, a proliferative stress such as chemotherapy is required to create a context in which Kmt2c deletion convey a selective advantage. We have also identified a mechanism, to explain this phenotype. Kmt2c encodes MLL3, a SET domain protein that binds enhancers and facilitates transcription. We have shown that Kmt2c/MLL3 deletions impairs enhancer recruitment during HSC differentiation, therefore blunting HSC commitment.Our findings suggest that acquired or pre-existing 7q (KMT2C) deletions may select for HSCs that could give rise to MDS/AML in the setting of autologous-transplantation. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that is often used to expedite neutrophil recovery after chemotherapy and to mobilize HSCs for collection and transplantation. We considered the possibility that 7q deletions, and KMT2C deletions in particular, may promote disproportionate mobilization of the mutant HSCs in response to G-CSF. To test this, we treated Kmt2c f/f; Vav1-Cre and Kmt2c f/+; Vav1-Cre mice with G-CSF, and we assessed HSC mobilization to the spleen and bone marrow. A far greater proportion of HSCs with heterozygous and homozygous Kmt2c deletions mobilized in response to G-CSF, relative to wild type HSCs. Kmt2c deletion also enhanced colony forming unit frequency in the peripheral blood after G-CSF treatment. Total body HSC numbers did not change in the body after G-CSF treatment on any genetic background, indicating that Kmt2c deletions enhanced HSC mobilization in response to G-CSF rather than self-renewal.To more faithfully recapitulate clinical conditions, we used Fgd5-CreER to delete a single Kmt2c allele in only a minority of HSCs. We then tested whether the mutant HSCs mobilized more efficiently than wild type HSCs. Surprisingly, Kmt2c deletions did not enhance HSC mobilization in this context. This raised the question of whether Kmt2c deletion in a non-HSC population could promote HSC mobilization in the Kmt2c f/+; Vav1-Cre mice. Indeed, analysis of mice chimeric for wild type and Kmt2c f/+; Vav1-Cre bone marrow suggested that Kmt2c non-cell autonomously regulates HSC mobilization. Finally, Kmt2c deletions did not enhance mobilization following exposure to plerixafor, a CXCR4 antagonist that acts directly on HSCs to promote mobilization rather than indirectly via monocyte populations, as occurs with G-CSF. Additional studies are needed to elucidate the mechanism by which Kmt2c non-cell autonomously regulates HSC mobilization. Our findings provide reassurance that, in a clinical setting, rare KMT2C-mutant HSCs will not disproportionately mobilize prior to apheresis. Furthermore, the data suggest that transient inhibition of MLL3, or its targets, may enhance HSC mobilization and negate selective advantages that 7q-deleted HSCs may acquire after chemotherapy treatment. DisclosuresNo relevant conflicts of interest to declare.

Altered HSC Programming Drives Aberrant Blood Output Underlying RA Pathogenesis

Rheumatoid arthritis (RA) is a debilitating autoimmune disorder characterized by autoantibody production towards collagen type-II in the synovial joints of the skeletal system, leading to progressive joint damage. RA is accompanied by chronic systemic production of pro-inflammatory cytokines including IL-6, TNF, and IL-1. Importantly, RA patients often suffer from significant hematological complications including production of tissue-damaging macrophages, chronic anemia, impaired lymphopoiesis and suppressed bone marrow function. As blood-forming hematopoietic stem cells (HSC) constitute the root of the immune system, identifying mechanisms by which altered HSC function may contribute to disease could provide key insights into RA pathogenesis and therapeutic intervention.In this study, we used a mouse model of collagen-induced arthritis (CIA), which faithfully recapitulates many features of human autoimmune arthritis, to identify the impact of disease on the hematopoietic compartment, and identify mechanisms underlying alteration in blood lineage output and function. Strikingly, bone marrow hematopoietic populations were significantly altered in CIA mice. This was typified by decreased numbers of peripheral red blood cells (RBC) and bone marrow erythroid progenitors, alongside decreases in bone marrow B cells and common lymphoid progenitors (CLP). On the other hand, we observed increased numbers of platelets, bone marrow granulocytes, and myeloid-biased multipotent progenitors (MPP3). Strikingly, we also observed reductions in the stromal cells comprising the bone marrow niche, with decreases in epithelial, mesenchymal, and osteoblastic cell types, suggesting that the inflammatory effects of RA influence the bone marrow compartment at multiple levels. We functionally validated our phenotypic analyses using colony forming unit (CFU) assays, and observed increased myeloid CFU, with concomitant decreases in erythroid and lymphoid colony forming activity in unfractionated bone marrow cells from CIA mice. Despite these changes, transplantation assays using purified HSCs from CIA and control mice showed that long-term reconstitution potential of HSCs was largely unaltered, suggesting that HSC self-renewal capacity is not significantly impaired. Taken together, these observations suggest that autoimmune arthritis induces substantial remodeling of the bone marrow, resulting in overproduction of myeloid cells at the expense of other lineages.In line with our phenotypic and functional analyses, RNA-seq analysis of HSCs from CIA versus control mice uncovered precocious activation of myeloid-specific gene programs that likely underlie shifts in lineage output. Moreover, in line with our transplantation data, we did not observe significant shifts in the expression of core genes governing HSC self-renewal. Collectively, these data show that the inflammatory environment of RA causes changes to hematopoietic output, leading to an increase in damaging myeloid cell types, further fueling the inflammatory environment and disease progression.We are currently identifying the mechanism underlying myeloid overproduction in the bone marrow, with focus on systemic inflammatory factors and/or shifts in the gastrointestinal microbiota, as potential underwriters of deregulated hematopoiesis and disease development. We are also assessing the functionality of myeloid cells generated by HSCs during RA to determine whether they exhibit a stronger pro-inflammatory phenotype. Ultimately, these studies will increase our understanding of the crosstalk between RA and the hematopoietic system, and potentially opens new avenues for therapeutic intervention to re-establish the hematopoietic system and even rescue RA progression. DisclosuresNo relevant conflicts of interest to declare.

MLL3 Deletion Enhances HSC Self-Renewal

Mixed lineage leukemia 3 (MLL3 ) is one of the most commonly mutated genes in human cancer, and it is believed to function as a tumor suppressor. It is one of many genes affected as a result of mono-allelic 7q deletions that occur commonly in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MLL3 encodes a histone methyltransferase that catalyzes the monomethylation of Histone 3 lysine 4 (H3K4), a known feature of enhancer elements. MLL3 also nucleates a COMPASS-like complex that removes repressive H3K27me3 marks, recruits the histone acetyltransferase p300/CBP to enhancers and promotes transcript elongation at promoters. For these reasons, MLL3 binding at cis-regulatory elements is thought to potentiate enhancer activity and transcription. Previous studies have shown that Mll3 deficiency causes a mild myeloproliferative disorder in mice (PLoS ONE 11:e0162515), and shRNA knockdown of Mll3 has been shown to cooperate with loss of Nf1 and p53 to promote AML (Cancer Cell 25:652). In the latter study, Mll3 silencing was associated with a hematopoietic stem cell (HSC)-like gene expression signature within the AML cells. These studies implicate MLL3 as a hematopoietic tumor suppressor, though its precise function in HSC self-renewal and lineage commitment has not been described.To better understand how Mll3 regulates HSC self-renewal, we developed a novel loss-of-function mouse allele in which a 5-base pair deletion and frameshift mutation was created near the N-terminus of the protein. Homozygous Mll3-/- mice are viable through most of gestation, but they die just prior to birth. Heterozygous Mll3+/- mice are born at normal Mendelian ratios, their development is normal after birth and they are fertile. To test whether Mll3 regulates hematopoiesis, we determined the frequencies and absolute numbers of HSCs, lineage restricted hematopoietic progenitors (HPCs), and granulocyte-monocyte progenitors (GMPs) in control, Mll3+/- and Mll3-/- fetal livers (E18.5) and control and Mll3+/- adult bone marrow. Heterozygous and homozygous Mll3 deletion caused a modest increase in HSC numbers and a modest decrease in HPC frequency in fetal livers. Mll3 heterozygosity resulted in a significant expansion of the HSC population in adult bone marrow. To test whether these increases in HSC numbers reflected enhanced self-renewal, we performed serial transplantation assays with fetal and adult HSCs. While primary transplants showed only a modest increase in repopulating activity in Mll3+/- and Mll3-/- HSCs, this effect was greatly enhanced in secondary transplants. Thus, Mll3 deficiency enhances HSC self-renewal capacity. Despite this phenotype, RNAseq showed no differences in the transcriptomes of wild type and Mll3+/- HSCs or HPCs. This suggests that Mll3 loss may reduce the probability of lineage commitment during successive HSC divisions without altering the commitment pathways themselves. Ongoing experiments will test this hypothesis. Enhanced self-renewal, particularly under conditions of hematopoietic stress, may convey a selective advantage to Mll3 -heterozygous HSCs and enhance progression to MDS. DisclosuresNo relevant conflicts of interest to declare.

Gain-of-Function of ASXL1 Truncating Protein in the Pathogenesis of Myeloid Malignancies

Introduction: Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We and others have reported that loss of ASXL1 impairs hematopoietic stem/progenitor cell functions (HSC/HPCs) and leads to myeloid dysplastic syndrome (MDS) in mice. It remains controversial whether truncating mutations in ASXL1 result in gain- or loss-of-function, or whether they confer dominant-negative activity in vivo . Answering this question is critical for determining how to target these mutations therapeutically.Methods: We generated a transgenic mouse model using HS321/45-vav vector that expresses the Asxl1 Y588X cDNA under the control of the Vav1 promoter. The transgene contains the entire coding region of mouse Asxl1 (full-length) with a stop codon mutation at the Y588 site (Asxl1 Y588X), leading to a production of truncating protein ASXL1aa1-587 in the hematopoietic system only. Utilizing the transgenic model, we performed a serial hematopoietic analyses, including peripheral blood (PB) counts, histological characterization on blood smear, tissue sections of femur and spleen, and flow cytometric analysis. Bone marrow(BM) transplantation was performed to identify the effects of truncating ASXL1aa1-587 on HSC/HPCs functions. RNA-seq and ATAC-seq analyses were performed to survey the differentially expressed genes in HSC/HPCs of WT and Asxl1 Y588X Tg mice. Liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation (IP) experiments were performed to identify the specific protein binding partner of ASXL1aa1-587. An epigenetic drug screening was used to identify specific targets to block the gain-of-function protein, ASXL1aa1-587, in the pathogenesis of myeloid malignancies in vivo.Results: The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patientswith ASXL1 mutations. Competitive transplantation of WT and Asxl1Y588XTg BM cells revealed that expression of A SXL 1 aa587 was associated with a higher percentage of CD45.2+ cells in the PB of the recipients, indicating that A SXL11-587aa expression increases HSC self-renewal capacity in vitro and in vivo. ATAC- and RNA-Seq analyses revealed that the ASXL11-587aa truncating protein expression results in more open chromatin in cKit+ cells compared to wildtype cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. LC-MS) and co-IP experiments showed that ASXL11-587aa acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hyper-sensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors.Conclusion: This study demonstrates that ASXL11-587aa plays a gain-of-function role in promoting myeloid malignancies and that BET bromodomain inhibitors significantly reduced the colony forming capacity of ASXL1Y588XTgHSPCs. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Inflammation, Aging and Hematopoiesis: A Complex Relationship.

All vertebrate blood cells descend from multipotent hematopoietic stem cells (HSCs), whose activity and differentiation depend on a complex and incompletely understood relationship with inflammatory signals. Although homeostatic levels of inflammatory signaling play an intricate role in HSC maintenance, activation, proliferation, and differentiation, acute or chronic exposure to inflammation can have deleterious effects on HSC function and self-renewal capacity, and bias their differentiation program. Increased levels of inflammatory signaling are observed during aging, affecting HSCs either directly or indirectly via the bone marrow niche and contributing to their loss of self-renewal capacity, diminished overall functionality, and myeloid differentiation skewing. These changes can have significant pathological consequences. Here, we provide an overview of the current literature on the complex interplay between HSCs and inflammatory signaling, and how this relationship contributes to age-related phenotypes. Understanding the mechanisms and outcomes of this interaction during different life stages will have significant implications in the modulation and restoration of the hematopoietic system in human disease, recovery from cancer and chemotherapeutic treatments, stem cell transplantation, and aging.

TWIST1 preserves hematopoietic stem cell function via the CACNA1B/Ca2+/mitochondria axis

AbstractMitochondria of hematopoietic stem cells (HSCs) play crucial roles in regulating cell fate and preserving HSC functionality and survival. However, the mechanism underlying HSC regulation remains poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulation of mitochondrial function. We demonstrate that Twist1 deletion results in significantly decreased lymphoid-biased HSC frequency, markedly reduced HSC dormancy and self-renewal capacity, and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient HSCs are more compromised in tolerance of irradiation- and 5-fluorouracil–induced stresses and exhibit typical phenotypes of senescence. Mechanistically, Twist1 deletion induces transactivation of voltage-gated calcium channel (VGCC) Cacna1b, which exhausts lymphoid-biased HSCs, impairs genotoxic hematopoietic recovery, and enhances mitochondrial calcium levels, metabolic activity, and reactive oxygen species production. Suppression of VGCC by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through the CACNA1B/Ca2+/mitochondria axis and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate.

Kmt2c mutations enhance HSC self-renewal capacity and convey a selective advantage after chemotherapy

SUMMARYThe myeloid tumor suppressor KMT2C is recurrently deleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), particularly therapy-related MDS/AML (t-MDS/t-AML), as part of larger chromosome 7 deletions. Here, we show that KMT2C deletions convey a selective advantage to hematopoietic stem cells (HSCs) after chemotherapy treatment that may precipitate t-MDS/t-AML. Kmt2c deletions markedly enhance murine HSC self-renewal capacity without altering proliferation rates. Haploid Kmt2c deletions convey a selective advantage only when HSCs are driven into cycle by a strong proliferative stimulus, such as chemotherapy. Cycling Kmt2c-deficient HSCs fail to differentiate appropriately, particularly in response to interleukin-1. Kmt2c deletions mitigate histone methylation/acetylation changes that accrue as HSCs cycle after chemotherapy, and they impair enhancer recruitment during HSC differentiation. These findings help explain why Kmt2c deletions are more common in t-MDS/t-AML than in de novo AML or clonal hematopoiesis: they selectively protect cycling HSCs from differentiation without inducing HSC proliferation themselves.

Fzr1 Preserves Hematopoietic Stem Cell Quiescence through Inhibiting Runx1

Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. FZR1, as a regulatory subunit of anaphase promoting complex/cyclosome (APC), is a master regulator of cell cycle, but little is known with regard to its role in HSC quiescence. Thus, we examined the function of Fzr1 in HSCs during steady-state hematopoiesis and under stress. We demonstrate that Fzr1 deletion led to perturbed hematopoiesis with an approximately 2-fold decrease in HSC pool size, as consequence of Fzr1 loss driving HSC from quiescence into rapid cycling (~70.1% to ~28.4%), elevating proliferation (~4.58% to ~18.5%) and apoptosis (~0.23% to ~2.86%) in HSC. As shown by serial bone marrow transplantation and competitive repopulation assays, self-renewal capacity and regenerative capacity were impaired, but differentiation of HSCs was not affected post Fzr1 deletion. Mechanistically, Fzr1 loss led to upregulation of Runx1 expression, in line with the protein expression, the ubiquitin sites of the gene was also obviouly decreased, knockdown Runx1 can rescue the Fzr1-deficient phenotype in HSC. Together, our data therefore support that Fzr1 acts a positive regulator of HSC quiescence and self-renewal capacity through inhibiting Runx1 expression. Disclosures No relevant conflicts of interest to declare.

3101 – EBF1 CONTRIBUTES TO THE SELF-RENEWAL CAPACITY AND LINEAGE DIFFERENTIATION OF HSPCS

Hematopoietic stem cells (HSCs) continually decide to either self-renew or differentiate. In this study we explore the role of lineage-specific transcription factors in HSC cell fate decisions, in particular Early B cell Factor 1 (EBF1) which orchestrates the B cell lineage program and is essential for B cell differentiation. Here, we use Ebf1flox/floxTie2Cre (Ebf1KO) mice to examine its potential role in hematopoietic stem and progenitor cells (HSPCs). We find that Ebf1KO mice display an increased number of HSCs that are less quiescent. Upon chronic 5-fluorouracil treatment, Ebf1KO mice show reduced survival as compared to wild type (WT) mice, suggesting that Ebf1 helps to maintain HSC quiescence. Additionally, competitive adoptive transfers of Ebf1KO HSCs into irradiated WT mice show a decrease in chimerism in both primary and secondary recipients compared to WT HSCs, indicating that Ebf1 is required for HSC self-renewal capacity. Ebf1KO mice also reveal a significant expansion of myeloid-biased (CD41+) HSCs, myeloid-biased multipotent progenitors (MPP2/3) and mature myeloid cells. B cell-deficient IgM-/- mice do not show an increase in HSC number, loss of quiescence nor loss of chimerism in adoptive transfer experiments, therefore the observed phenotypes in Ebf1KO mice are specific to Ebf1 and a secondary effect of the loss of B cells. Additionally, we find that Ebf1KO HSPCs are not marked by DJ recombination demonstrating that the phenotypes of Ebf1 KO mice are not due to the dedifferentiation of B cells. These data indicate that the loss of Ebf1 in HSPCs results in a myeloid-biased output and that Ebf1 has an unexpected additional function in the regulation of HSC self-renewal and differentiation.

Short-term ex-vivo exposure to hydrogen sulfide enhances murine hematopoietic stem and progenitor cell migration, homing, and proliferation

ABSTRACT For successful transplantation of Hematopoietic Stem cells (HSCs), it is quite necessary that efficient homing, engraftment and retention of HSC self-renewal capacity takes place, which is often restricted due to inadequate number of adult HSCs. Here, we report that short-term ex-vivo treatment of mouse bone marrow mononuclear cells (BMMNCs) to Sodium Hydrogen Sulfide (NaHS, hydrogen sulfide-H2S donor) can be used as a possible strategy to overcome such hurdle. H2S increases the expression of CXCR4 on HSPCs, enhancing their migration toward SDF-1α in-vitro and thus homing to BM niche. . Additionally, in-vitro studies revealed that H2S has a role in activating mitochondria, thus, pushing quiescent HSCs into division. These results suggest a readily available and cost-effective method to facilitate efficient HSC transplantation.

Kmt2c Limits the Self-Renewal Capacity of Multiply Divided HSCs By Promoting Sensitivity to Interleukin-1

Under normal homeostatic conditions, adult hematopoietic stem cells (HSCs) are usually quiescent. Hematopoietic stress, such as blood loss, infection, inflammation, or chemotherapy, can drive HSCs into cycle. When adult HSCs divide multiple times, they lose self-renewal capacity. Inflammatory cytokines, such as interleukin-1 (IL-1), can accelerate the loss of HSC self-renewal capacity by activating PU.1 and promoting myeloid commitment. This raises the question of whether intrinsic tumor suppressor genes can modulate sensitivity to inflammatory cytokines, and whether loss of these tumor suppressors can allow HSCs to evade commitment programs that would otherwise limit self-renewal capacity. The KMT2C tumor suppressor is located on chromosome 7q within a region that is frequently deleted in myelodsplastic syndrome (MDS) and therapy-related acute myeloid leukemia (AML). It encodes MLL3, a histone methyltransferase that activates enhancer elements and promotes transcription. Haploid KMT2C deletion has previously been shown to activate self-renewal programs and accelerate AML formation. This raised the question of whether KMT2C/MLL3 regulates normal HSC self-renewal and whether KMT2C deletion conveys a selective advantage to HSCs in contexts that would otherwise deplete the HSC pool. By protecting HSCs from exhaustion, KMT2C deletions may indirectly facilitate 7q-deficient MDS/AML. To understand whether and how Kmt2c regulates HSC self-renewal, we developed novel germline and conditional Kmt2c knockout mouse alleles. Mono- and bi-allelic Kmt2c deletions led to a modest increase in adult HSC numbers and a significant reduction in committed hematopoietic progenitors (HPCs). Kmt2c deletions markedly enhanced HSC self-renewal capacity, but HSC proliferation rates were not altered. To mimic conditions that lead to therapy-related AML, we deleted a single Kmt2c allele in a minority of HSCs. We then tested whether the Kmt2c-deleted HSC population expanded, relative to wild type HSCs, under native and stressed conditions. Under native conditions, the percentage of Kmt2c-heterozygous HSCs remained stable. However, after several cycles of chemotherapy, the Kmt2c mutant HSCs predominated within the marrow. In mechanistic studies, RNA-sequencing showed that Kmt2c-deficient HSCs expressed genes associated with innate immune signaling, including the receptor for interleukin-1 (IL1R), at lower levels than wild type HSCs. This suggested that Kmt2c mutations might sustain self-renewal capacity in multiply divided HSCs by dampening IL-1 driven myeloid commitment. In support of this hypothesis, Kmt2c-deficient HSCs retained multilineage potential when they were cultured with IL-1, and they failed to activate JNK and p38 upon exposure to IL-1. Altogether, our data suggest a mechanism to explain how KMT2C deletions, in the context of larger 7q deletions, may promote therapy related MDS/AML. When HSCs acquire a KMT2C deletion, they can then escape IL-1-mediated exhaustion when they are driven into cycle by chemotherapy or other stressors. In lieu of chemotherapy-induced stress, the same clones may remain relatively indolent. Disclosures No relevant conflicts of interest to declare.

Ripk3 Signaling Regulates Hematopoietic Stem Cell Number and Function during Stress

Background: Among all tissues, bone marrow (BM) is the most sensitive tissue to ionizing radiation (IR)-induced acute tissue damage (ATD) and chronic long-term residual damage (LT-RD). BM failure and a significant reduction in blood cells (pancytopenia) often occurs within days after exposure to IR due to the massive death of proliferative hematopoietic progenitor cells (HPCs). However, due to their quiescent cell cycle status and reduced fidelity of DNA repair feature, many hematopoietic stem cells (HSCs) cannot fully eliminate such damage and enter senescence; this results in LT-RD. Abnormal dysplastic hematopoiesis is the most common LT-RD in most victims of IR, followed by an increased risk of leukemia/lymphoma development. Thus IR exposure is an established cause of BM failure and leukemia. A significant increase in the production of inflammatory cytokines is induced by IR which contributes to the pathogenesis of both ATD and LT-RD. Such inflammatory cytokines induce the activation of Ripk3-Mlkl-mediated necroptotic signaling in HSCs. However, the role of Ripk3-Mlkl signaling in IR-induced damage has not studied. Experimental procedures: The self-renewal capacity of HSCs among Ripk3-/-, Mlkl-/- and WT mice were examined and compared by serial transplantation assay. The phenotypes of ATD and LT-RD induced by different dosages of IR were compared among Ripk3-/-, Mlkl-/- and WT mice. The mechanism by which Ripk3 signaling prevents IR-induced leukemia development was studied. Results: Ripk3-Mlkl signaling is not required for hematopoiesis during homeostatic condition. However, during serial transplantation, inactivation of such signaling prevents stress-induced loss of HSCs. Interestingly, Ripk3 signaling also induces an Mlkl-independent ROS-p38-p16-mediated senescence in HSCs. Thus Ripk3-/- HSCs showed better competitive hematopoietic ability compared to Mlkl-/- and WT HSCs during serial transplantation. A sub-lethal dosage of IR (6Gy) induces Ripk3-dependent NF-κB activation and pro-survival gene expression in HSCs, which is necessary for the survival of damaged HSCs. After 6Gy IR, although DNA damage is repaired in most HSCs within 2 days, a proportion of HSCs in WT and Mlkl-/- mice fail to fully repair the damage and undergo p53-p21-dependent senescence. However such cells in Ripk3-/- mice die from apoptosis. Thus the remaining HSCs in Ripk3-/- mice should be functionally normal, while a proportion of the remaining HSCs in Mlkl-/- and WT mice remain damaged but senescent, all as demonstrated by competitive hematopoietic reconstitution assay. Multiple low-doses of IR (1.75Gy once week × 4) induce HSC exhaustion in WT mice but not in Ripk3-/- and Mlkl-/- mice. Interestingly, almost all Ripk3-/- mice develop acute lymphoblastic leukemia within 200 days after such low dose IR, while 45% of WT and 60% of Mlkl-/- mice develop thymomas within 360 days (see Figure). Mechanistically, such low-dose IR stimulates chronic inflammatory cytokine production. Such cytokines induce Ripk3-Mlkl-mediated necroptosis in response to HSC exhaustion observed in WT mice. These cytokines also induce Ripk3-ROS-p38-p16-mediated senescence in response to impaired HSC functioning observed in both WT and Mlkl-/- mice. In Ripk3-/- mice, due to the lack of both necroptotic and senescent signaling, mutant HSCs accumulate and leukemia development is accelerated. Conclusion: Ripk3 signaling plays distinct roles in HSCs in response to different doses of IR. High-dose IR induces Ripk3-dependent NF-κB/survival signaling, which is required for the survival of HSCs which fail to repair the damage. Thus temporal inhibition of Ripk3-NF-κB signaling might help to remove the damaged HSCs thus preventing the occurrence of LT-RD. However multiple low-doses of IR induces Ripk3 activation in HSCs which represses leukemia development by inducing both ROS-p38-p16-mediated senescence and Ripk3-Mlkl-mediated necroptosis. Induced activation of Mlkl-necroptosis might help to repress leukemia development by removing damaged HSCs. Disclosures No relevant conflicts of interest to declare.

2001 - LONG-TERM EX VIVO EXPANSION OF FUNCTIONAL HEMATOPOIETIC STEM CELLS

The self-renewal of multipotent hematopoietic stem cells (HSCs) is key for life-long maintenance of the blood system and the curative capacity of clinical HSC transplantation. However, while in vivo HSC self-renewal capacity has been well-described, existing culture conditions poorly support ex vivo HSC self-renewal and afford only a very limited window to study or modify HSCs in-a-dish. By taking a reductionist optimization approach, we have developed a simple culture platform that supports functional mouse HSCs ex vivo over 1-2 months. Importantly, we identified the synthetic polymer polyvinyl alcohol as a superior, inexpensive, and chemically-defined alternative to serum albumin supplements, which have long represented a major source of biological contaminants and batch-to-batch variability in HSC cultures. Limiting dilution transplantation analysis of day-28 HSC cultures estimated a ∼900-fold expansion of functional HSCs (based on >1% multilineage engraftment at 16-weeks post-transplantation) with secondary transplantation analysis estimating >200-fold expansion of serially-engraftable long-term HSCs. HSCs could also be expanded clonally using this system, demonstrating bona fide ex vivo HSC self-renewal. The large numbers of functional HSCs generated by this long-term ex vivo expansion protocol even enabled HSC transplantation in nonconditioned immunocompetent and immunodeficient recipients. Finally, this albumin-free and GMP-compatible culture system also supported human HSCs ex vivo, as determined by long-term engraftment in NSG mice. Thus, our novel HSC culture platform provides an important new tool to interrogate HSC self-renewal and lineage commitment, and also suggests a novel approach in clinical HSC transplantation.

Epidermal Growth Factor Rejuvenates Aging Hematopoietic Stem Cells

Aging hematopoietic stem cells display distinct abnormalities such as myeloid skewing, decreased repopulating capacity, and leukemia predisposition. Radiation exposure potentiates aging of the hematopoietic system, as evidenced by premature and persistent senescence of HSCs in mice exposed to moderate dose irradiation (Blood, 2014. 123(20): p. 3105-15). Our laboratory demonstrated that EGF promotes hematopoietic stem cell (HSC) regeneration after radiation injury (Nat Med, 2013. 19(3): p. 295-304). Based on these results, we hypothesized that EGF and EGFR signaling may have rejuvenating effects on aging HSCs.For comparisons of young and aged mice, we utilized 2-4 month old mice and >18-24 month old female C57BL/6 mice, respectively. We discovered that aged C57BL/6 mice have decreased levels of EGF in the peripheral blood compared to young mice (p= 0.02) and decreased expression of EGFR on bone marrow (BM) ckit+sca-1+lin- (KSL) stem/progenitor cells (p= 0.03). BM KSL cells from aged mice displayed increased DNA damage in culture with thrombopoietin, SCF and Flt-3 ligand (TSF) compared to young KSL cells (p< 0.0001). Aged BM KSL cells treated with 100 ng/ml EGF had decreased DNA damage compared to aged BM KSL cells treated with TSF only (p< 0.01) Treatment with EGF for 7 days increased the colony forming capacity of aged BM KSL cells (p< 0.01). Importantly, competitive repopulation assays revealed that 7 day in vitro treatment of aged BM KSL cells with EGF significantly increased primary donor cell engraftment (p< 0.01) at 16 weeks compared to aged BM KSL cells cultured with TSF alone.Based on these in vitro observations, we next tested whether systemic administration of a recombinant, pegylated EGF (pEGF), 20 mcg three times weekly for 4 weeks could alter the hematopoietic characteristics of aged C57BL/6 mice. pEGF treatment decreased BM myeloid skewing (p< 0.01) and increased CD3 T cell content (p< 0.01) in aged mice compared to saline-injected controls. Furthermore, competitive repopulation assays revealed that pEGF treatment of aged mice caused a significant increase in functional HSCs capable of competitive engraftment in recipient congenic mice. Total primary donor cell (p< 0.01), myeloid cell (p< 0.01), T cell (p< 0.01) and B cell (p= 0.01) engraftment were significantly increased in recipient mice transplanted with BM cells from aged, pEGF-treated donors compared to recipients of aged, saline-treated donors at 20 weeks. Additionally, secondary transplantation also resulted in increased donor engraftment (p< 0.01) and increased donor myeloid engraftment (p< 0.05) at 12 weeks. These results suggested that systemic administration of pEGF rejuvenates the repopulating ability of aged HSCs.We next sought to determine if deficiency in EGFR signaling could accelerate hematopoietic aging in mice. For this purpose, we utilized a doxycycline-inducible, hematopoietic cell specific EGFR dominant negative mutant model (SCL-tTA;EGFR-DN mice). Interestingly, adult (13 month old) SCL-tTA;EGFR-DN mice displayed increased myeloid skewing in the peripheral blood (p< 0.05) and BM cells from SCL-tTA;EGFR-DN mice generated significantly decreased colony forming cells, compared to age-matched, EGFR-expressing mice (p< 0.01). Mechanistically, BM KSL cells from 13 month old SCL-tTA;EGFR-DN mice demonstrated increased senescence (p< 0.01) and increased expression of p16 (p< 0.05) compared to young mice. In contrast, BM KSL cells from aged C57BL/6 mice that were treated with 100 ng/ml EGF (plus TSF) for 7 days displayed decreased senescence and decreased p16 expression compared to BM KSL cells from aged mice treated with TSF alone. Taken together, these studies suggest that EGF/EGFR signaling declines with age and that reactivation of EGFR signaling via EGF treatment can ameliorate clinically relevant features of hematopoietic aging, including HSC self-renewal capacity. DisclosuresNo relevant conflicts of interest to declare.